Abnormalities in potassium and acid-base balance are often serious clinical challenges in critically ill patients. The kidney plays a decisive role in maintaining total body potassium balance through regulation of reabsorptive and secretory processes involving activity of K+-specific cation channels and ATP-driven cation pumps. Two H+, K+-ATPases have been localized to kidney and have been shown to participate in K+ absorption and urinary acidification. Previous studies in our laboratory have shown that the colonic alpha-H+, K+-ATPase (HKalpha2) subunit is up regulated by chronic hypokalemia. Others have shown up regulation by metabolic acidosis, metabolic alkalosis, salt depletion, and in the developing kidney. This regulatory response is confined primarily to the outer and inner medullary-collecting duct. A challenge in the field has been to reconcile the divergence in functional properties observed in vitro with expressed function. Three strategic observations during the last funding cycle made by our laboratory include: 1) that the association of HKalpha2 in vivo with the beta1-Na+, K+-ATPase subunit protects the complex from degradation and fosters translocation to the plasma membrane, 2) that a segment of the carboxy terminal of HKalpha2 is essential for HKalpha2 function, and 3) that the HKalpha2/beta1 complex associates with the tetraspanin protein CD63. Therefore, over the next five years we will investigate: 1) the biologic role of specific regions of the carboxy-terminus of HKalpha2 and other X+, K+-ATPases, and 2) the physiological role of the association with CD63. Accordingly, three Specific Aims will be addressed in the next period of this award: Aim 1 will characterize the physiological significance of our observation of unique association between the carboxy-terminus of HKalpha2 and CD63. Aim 2 will identify and characterize the specific role of the carboxy-terminus of HKalpha2 in the biological activity of the colonic H+, K+-ATPase. Aim 3 will investigate trafficking and translocation of the HKalpha2/beta1/CD63 complex to the plasma membrane. The overall objective of research by this laboratory is to clarify the molecular basis of responses by the kidney to regulate potassium and acid-base homeostasis. This research will advance knowledge of fundamental mechanisms of potassium and acid base balance in health and models of common clinical circumstances associated with deranged electrolyte balance.